Despite the human cost of tuberculosis (TB), the disease remains difficult to diagnose and thus to control. One-third of the world's population is infected with M. tuberculosis. Every year, more than 9 million people develop active TB and 1.7 million people die from the disease [WHO 2010]. Current widely used diagnostics perform poorly in identifying active TB in patients with roughly 50% of cases having sputa that are smear negative for acid fast bacilli (AFB) [Steingart 2006]. Undiagnosed cases unwittingly continue to transmit disease, threatening public health. In the US, the uncertainty in diagnosis, and the absence of sensitive, specific, and rapid tests, results in significant increases in hospitalizatio costs because Centers for Disease Control and Prevention (CDC) guidelines recommend that suspected cases of TB be isolated in single bed, negative-pressure rooms until three consecutive daily sputum samples have been processed by direct smear and deemed negative for AFB [CDC 2005]. Due to logistics issues, these patients remain in isolation an average of five days before being cleared. Because the vast majority of these patients do not have the disease, TB control cost at the inpatient level is dominated by these inpatient isolation days for TB suspects [Scott 1994] and is often ultimately borne by the hospital itself. Compounding this infection control approach is the fact that negative sputum smears for AFB do not definitively exclude risk of TB transmission [Behr 1999]. Societal costs increase dramatically when undiagnosed cases continue to transmit disease [Miller 2010]. The limitations of the current gold standards for TB diagnosis have stimulated renewed interest in rapid, accurate, cost-effective, non-invasive, and user-friendly methods of identifying M. tuberculosis from TB suspects. Patient cough aerosol analysis for M. tuberculosis has the potential to address all these requirements. Livermore Instruments' unique Single Particle Aerosol Mass Spectrometry (SPAMS) technology may be the answer to TB diagnosis in clinical real-world settings. SPAMS has been previously deployed by the Department of Energy for the detection of B. anthracis. In this application, we describe an aerosol analysis system based on SPAMS as a potential tool for detection of M. tuberculosis in coughed and exhaled breath. The existing versions of our system are simple to operate, sturdy, contain few moving parts, and, importantly, are reagent-free so that the marginal cost per test is extremely low. SPAMS functions by drawing in individual aerosol particles and tracking them with lasers as they proceed towards the center of the source region of a dual-polarity time-of-flight mass spectrometer. Upon their arrival, they are desorbed and ionized by a single, pulsed high power laser and the resulting ions are measured by the mass spectrometer. Real-time analysis of the mass signature allows the identification of the particles. In the case of many microorganisms, they can be identified to the species level. A SPAMS system can evaluate dozens to hundreds of particles per second, collecting 1000 mass spectra, each of an individual microorganism, in 90 seconds. In this application, we describe the evaluation of SPAMS for the detection of M. tuberculosis from coughed aerosol of TB suspects. M. tuberculosis has been detected in the aerosol phase in clinical settings previously but such detection was performed labor-intensively using the polymerase chain reaction (PCR) to analyze samples collected over a matter of hours [Chen 2005]. We will prove that the diagnosis of TB in real- time is a viable concept by rapidly and accurately detecting M. tuberculosis using this state-of-the-art technology. This is a transformative concept with many potential applications, including rapid triage in hospitals at the emergency room or at TB control clinics to identify patients in need of treatment and to prevent nosocomial transmission, as well as for rapid mass screening in non-clinical environments such as in the field in high incidence settings. Finally, another key advantage of this technology that distinguishes it from other breath analysis systems under development is the ability to train SPAMS for the detection of other respiratory pathogens as well. This makes SPAMS ideal as a diagnostic in TB patients in whom parenchymal damage and bronchiectasis often result in concomitant infection/colonization with other non-TB pathogens. The ability to diagnose other respiratory diseases further broadens SPAMS' potential use in a variety of clinical and non-clinical settings. Livermore Instruments Inc. is a San Francisco Bay Area startup company dedicated to bringing aerosol analysis to nontraditional fields.